RESUMEN
Cryptophytes are among the few eukaryotes employing phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to cyanobacterial PBP that are organized in membrane-associated phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin 545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. The assembly of PBPs in cryptophytes involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of the eukaryotic S-type PBP lyase GtCPES. We show GtCPES-mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP ß-subunit (PmCpeB) of Prochlorococcus marinus MED4. On the basis of the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB), most likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colored complex in vitro and produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on these findings, we postulate that this single amino acid residue has a crucial role for bilin binding specificity of S-type phycoerythrin lyases but additional factors regulate handover to the target protein.